A major goal of modern Neuroscience is to understand the neuronal basis of behavior. Locomotion is a behavior generated in the spinal cord by complex neuronal circuits. It is defined as precise, coordinated and alternating activity between opposing limbs as well as between antagonistic muscles of the same limb. Furthermore, locomotor behavior is attractive for experimental study because it can be easily accessed, defined, and quantified. A network of interneurons, known as the central pattern generator (CPG), is thought to be responsible for the genesis of rhythmic activity. CPG neurons activate motor neurons which in turn activate peripheral muscles resulting in movement. Motor neurons also possess axon collaterals which target exclusively a class of inhibitory interneurons, known as Renshaw cells. Our recent studies have challenged the traditional idea that motor neurons are simply the motor output from the spinal cord. Stimulation of motor neuron axons in neonatal mice can trigger locomotor activity in the presence of cholinergic receptor antagonists. This suggests that acetylcholine released by motor neuron axon collaterals is not required for rhythmogenesis. Furthermore, we discovered that neonatal motor neurons release a second fast excitatory neurotransmitter from their axon collaterals (glutamate or aspartate) in addition to acetylcholine. The mechanisms of how stimulation of motor axons can trigger locomotor-like activity in neonatal spinal cords are not understood. The identification of neurons and their connectivity that participate in the locomotor CPG is critically needed for the elucidation of mechanisms involved in locomotor activity and would therefore represent a fundamental advance in the field. In this proposal, we provide some preliminary evidence that a novel class of excitatory interneurons is a critical component in a ventral spinal cord circuit, putatively connected to lumbar motor neurons and it may be involved in the locomotor central pattern generator. We have designed a set of experiments to test our main hypothesis, that motor neurons play an active role in the generation of locomotor activity. In Aim 1, we will attempt to identify the neuronal targets of this novel type of interneuron using physiological and morphological assays. In addition, we will investigate the participation of these interneurons in locomotor-like behavior using modern optophysiological methods employing the in vitro spinal cord preparation. Optophysiological approaches combine optical imaging with electrophysiological techniques. In Aim 2, we will perform laser capture microdissection from these interneurons compared to motor neurons using a differential gene microarray analysis. Validation of gene(s) expressed solely in these interneurons will be performed by in situ hybridization techniques. In summary, this two-year research project describes a comprehensive set of experiments that have the potential to identify a novel class of excitatory interneurons as a key neuronal player in the rhythmogenesis of locomotor activity.